The major blood-clotting-protein, fibrinogen, is synthesized in the liver and secreted into the bloodstream as a multimer of three different subunits, designated Aalpha, Bbeta, and gamma. The overall goal of the research described in this proposal is to determine how expression of the separate genes coding for the three fibrinogen subunits is coordinately regulated. The basic experimental strategy is to use the powerful gene transfection approach to generate precise mutations in the fibrinogen gene regulatory regions in vitro and analyze the functional consequences of these alterations after introducing the DNA into cultured hepatocytes. Highly purified hepatocytes from the frog Xenopus laevis will be used, in which fibrinogen synthesis is dramatically induced by the adrenal steroids, glucocorticoids. This proposal focuses specifically on the gene coding for the gamma subunit, with the following specific aims; 1) Isolation of the upstream regulatory region of the gamma fibrinogen subunit gene; 2) Introduction of the gamma gene DNA into primary Xenopus liver cells by transfection and demonstration of glucocorticoid-inducible transcription; 3) Generation of deletion and point mutations in the transcriptional regulatory region and analysis of effects on transcription in transfected liver cells; 4) Identification of protein interactions with critical DNA regulatory elements; and 50 Development of an in vitro transcription system for dissecting the specific molecular interactions underlying control of transcription.